Frozen tissue sections
From Diseasepedia.net
No 3. Frozen tissue sections.Detection limit is approximately 1,000-10,000 molecules/cell if localized.
Detects antigen presence and localization
Qualititive to semi-quantitative
Sensitivity dependent on minimal level and localuzation of antigen
High local concentration of antigen, so lower-affinity antibody
Cautions
Diaminobenzidine (DAB), DPX, ethanol, hydrogen peroxide, nickel chloride, pataformaldehyde, sodium azide
1. Dissect a small sample. no bigger than approximately 1 cm2 * 0.4 cm, of undamaged tissue.
2. Place the specimen on one end of a strip of card. Label the card, and submerge the end with the sample in liquid nitrogen. After 60 seconds in the liquid nitrogen, remove the sample and place on dry ice. Trim the card to just larger than size of the tissue and transfer to a precooled (-70°C), labeled freezing vial.
3. Prior to sectioning, coat clean glass slides with 1% gelatin. Dissolve gelatin in water by heating to 50°C, cool, and add sodium azide to 0.02%. Dip the slides in the solution for 30 seconds, remove, and allow to air-dry.
4. Prepare sections of frozen tissues using a cryostat followinf the manufacturer's direcions. Sections between 5 um and 10 um commonly are used. Collect the sections on the coated slides.
5. Allow the sections to air-dry. Dip in freshly prepared 4% paraformaldehyde for 2 minutes.
6. Wash with several changes of PBS, and place in 1% NP-40, PBS, for 5 minutes. Wash with several changes of PBS.
7. Place slides with the tissue sections in a humidified chamber. Add the first antibody solution. All dilutions must be carried out in protein-containing solutions such as 3% BSA in PBS.
Monoclonalantibodies are best applied as tissue-culture supernatants (specific antibody concentration of 20-50 ug/ml, use neat). Ascites fluids, purified monoclonal and polyclonal antibodies, and crude concentrations between 0.1 and 10 ug/ml. If the specific antibody concentration of the antibody sample is unknownm prepare and test 1/10, 1/100, 1/1000, 1/10000 dilutions of the starting material.
8. Incubate the slides for a minimum of 60 minutes at room temperature in the humidified chamber. For some reactions, prolonged incubations of up to 24 hours can increase sensitivity.
9. Wash in three changes of PBS over 5 minutes.
10. Apply the horeseradish perixidase-labeled secondary reagent specific for your promary antibody. These reagents can be purchased from several suppliers.
If the correct amount of secondary reagent is not known, test dilutions of the secondary antibodies 1/50 to 1/1000. Carry out all dilutions in a protein-containing solution such as 3% BSA/PBS.
11. Incubate with the labeled secondary reagent for 30 minutes at room temperatur in the humidified chamber.
12. Wash in three changes of PBS over 5 minutes.
13. Prepare the DAB/metal reagent. Dissolve 6 mg of DAB in 9ml of 0.05M Tris buffer (pH 7.6). Add 1 ml of a 0.3% wt/vol stock solution of nickel chloride inwater (the same amount of cobalt chloride can be used as an alternative). Add 0.1 ml of a 3% solution of hydrogen peroxide i water. If a precipitate appears, dilter through Whatman No. 1 filter paper (or equivalent).
14. Apply the solution to the specimen. Observe the sample inder a low-power light microscope. When the bown/ black precipatate has developed sufficiently, stop the reaction by washing in water. This will normally be between 1 and 20 minutes.
15. Add a few drops of Harris' Hematoxylin to the specimen. Incubate for approximately 5 minutes. The length of time will determine the intesnsity of the stain.
16. Wash the slide gently in water.
17. Dehtdrate the sample by passing through graded alcohols. Incubate twince for 3 minutes each in 75% ethanol, twice for 3 minutes each in 95% ethanol, and twice for 3 minutes each in absolute ethanol. Air-dry.
18. Add a small drop of DPX to the specimen. Carefully place a coverslip (#1) on the drop, avoiding air bubbles. Remove any excess mounting medium with a paper towel.
DPX will set almost immediately and the samples can be observed and photographed.
